Magnetic resonance imaging (MRI) is a nuclear magnetic resonance technique that is used clinically to distinguish between different tissues or organs in the human or animal body through the spatial localization of water protons in the tissues or organs. The signal that is obtained by this technique and is then converted into an imaging depends in fact on the water proton concentrations and on the relaxation rates within the different types of tissues.
MRI often requires the use of contrast agents, i.e. agents that influence the local relaxation behaviour of the observed nuclei in certain tissues or organs, because if MRI is performed without employing a contrast agent, differentiation of the tissue of interest from the surrounding tissues in the resulting image may be difficult.
The in vivo utilization of paramagnetic complexes as non-specific agents for contrast enhanced MRI has been the subject of a number of different studies. Paramagnetic contrast agents involve materials which contain unpaired electrons. The unpaired electrons act as small magnets within the main magnetic field to increase the rate of longitudinal (T1) and transverse (T2) relaxation. Generally, paramagnetic contrast agents are used for their ability to decrease T1 (positive contrast agents) and in use they enhance image intensity from the regions to which they distribute.
Paramagnetic contrast agents typically comprise metal ions, such as for instance transition metal ions, which provide a source of unpaired electrons. Particularly preferred and therefore studied in more depth, resulted to be Gd3+ (with 7 unpaired electrons) and Mn2+ (with 5 unpaired electrons). Particular attention has been paid to the Gd3+ ion as this ion shows a very high magnetic moment coupled to a relaxation rate relatively long at the magnetic fields of interest in the MR area (in the range of nanoseconds). However these metal ions are also generally highly toxic or at least poorly tolerated and need to be strongly coordinated with a ligand that occupies the major number of coordination sites. Generally speaking, to control toxicity and at the same time get a sufficient contrast in the imaging, it is necessary to have paramagnetic complexes endowed with high thermodynamic and kinetic stability, containing at least one molecule of water directly coordinated to the metal ion in rapid exchange with the “bulk” water.
The first contrast agent for MRI approved by the Regulatory Authorities was GdDTPA (Magnevist®, by Schering AG), followed by GdDOTA (Dotarem®, by Guerbet SA), GdDTPA-BMA (Omniscan®, by Nycomed Imaging AS), and GdHPDO3A (ProHance®, by Bracco Imaging S.p.A.). The chemical formula of these contrast agents is reported hereinbelow

These four contrast agents share some similar pharmacological features as they all diffuse from plasma into the extracellular fluids and are excreted through the kidney via glomerular filtration. They are particularly useful for the diagnosis of hematoencephalic barrier lesions.
Linked thereto are other two Gd(III) complexes that are used in the imaging of the liver: Gd EOB-DTPA (Eovist®, by Shering AG) and Gd BOPTA (MultiHance®, by Bracco Imaging S.p.A.) (the chemical formula of the two ligands, EOB-DTPA and BOPTA, is reported below)

These two compounds are characterised by an increased lipophilic behaviour due to the introduction of an aromatic substituent in the ligand structure and for this reason are preferably uptaken by the liver cells.
Another class of compounds useful as contrast agents for MRI are ferromagnetic materials which are employed for their ability to decrease T2. Ferromagnetic materials have high, positive magnetic susceptibilities and maintain their magnetism in the absence of an applied field. Ferromagnetic materials for use as MRI contrast agents are for instance described in WO 86/01112 and WO 85/043301.
A third class of magnetic materials that can be used in MRI are those generally indicated as superparamagnetic materials. Analogously to paramagnetic materials, the superparamagnetic ones do not maintain their magnetism in the absence of an externally applied magnetic field. Superparamagnetic materials can have magnetic susceptibilities nearly as high as ferromagnetic materials and higher than the paramagnetic ones. As generally used, also superparamagnetic materials alter the MR image by decreasing T2 and therefore result in a darkening of the tissues or fluids where they are present or accumulate versus the lighter background where they are not present.
Iron oxides such as magnetite and gamma ferric oxide exhibit ferrromagnetism or superparamagnetism depending on the size of the crystals comprising the material, with larger crystals (typically with an average size larger than 0.3 μm) being ferromagnetic.
The general idea of an MRI enhancing contrast agent comprising a moiety that is detectable in a magnetic resonance imaging procedure linked to a molecule capable of specific binding to a cellular receptor is already known.
See for instance U.S. Pat. No. 4,827,945 that discloses i.e. coated magnetite particles for use as MRI contrast agents, said particles being surrounded by a polymer to which biologically active molecules, chosen to target specific organs or tissues, may be attached.
See also WO 01/30398 and the literature cited therein where the use of receptor-binding ligands bound to a paramagnetic chelate is described.
The idea is that since there are specific receptors which are known to be overexpressed in the cells of certain tumors, being able to selectively distinguish a tumor cell from a normal cell will allow to visualize and identify precise locations of the tumor masses and better manage the disease.
The paramagnetic, ferromagnetic or superparamagnetic compounds containing the suitably selected targeting moiety (e.g. antibody, antibody fragment, peptide, protein, and the like) bind to the relevant receptors on the surface of the tumor cells to be targeted and is possibly internalised. The number of receptors per cell however is generally lower than the number of MRI detectable metal ions required to have a MR signal visible with the actual MRI technologies. To increase the contrast and make the signal more visible it is therefore necessary to increase the concentration of the contrast agent in the cell or on the surface of the cell. This can be obtained either giving to the cell sufficient time to internalize the label/targeting compound (so that the signal can be given by the contrast agent inside the cell as well as around or on the surface of the cell) and/or administering simultaneously a compound capable to increase internalization of the receptor-binding compound, or increasing the number of MRI detectable metal units linked to the targeting moiety (via dendrimers or multimers). In the latter case, particularly useful is the internalization route based on receptor mediated (fluid) endocytosis that allows the entrapment of a huge number of paramagnetic units.
While theoretically excellent, in practice neither of these approaches has led to a commercial product yet. On the one hand, in fact, once the targeting MRI detectable compound is bound to the cell surface, it is not always possible to achieve or induce the desired internalization and on the other hand the use of large multimers is generally connected with an unacceptable increase in toxicity of the product because of the large molecular weight thus obtained.
For utility in diagnostic imaging the optimum contrast agent should provide a contrast sufficient to clearly distinguish between normal, healthy cells and tumor cells using the available equipment and without creating any toxicity problem.